Herbert B. Schiller

Quantitative proteomics of the integrin adhesome show a myosin II-dependent recruitment of LIM domain proteins.

A characteristic of integrins is their ability to transfer chemical and mechanical signals across the plasma membrane. Force generated by myosin II makes cells able to sense substrate stiffness and induce maturation of nascent adhesions into focal adhesions. In this paper, we present a comprehensive proteomic analysis of nascent and mature adhesions. The purification of integrin adhesion complexes combined with quantitative mass spectrometry enabled the identification and quantification of known and new adhesion‐associated proteins. Furthermore, blocking adhesion maturation with the myosin II inhibitor blebbistatin markedly impaired the recruitment of LIM domain proteins to integrin adhesion sites. This suggests a common recruitment mechanism for a whole class of adhesion‐associated proteins, involving myosin II and the zinc‐finger‐type LIM domain.

Mechanosensitivity and compositional dynamics of cell–matrix adhesions

Cells perceive information about the biochemical and biophysical properties of their tissue microenvironment through integrin‐mediated cell–matrix adhesions, which connect the cytoskeleton with the extracellular matrix and thereby allow cohesion and long‐range mechanical connections within tissues. The formation of cell–matrix adhesions and integrin signalling involves the dynamic recruitment and assembly of an inventory of proteins, collectively termed the ‘adhesome’, at the adhesive site. The recruitment of some adhesome proteins, most notably the Lin11‐, Isl1‐ and Mec3‐domain‐containing proteins, depends on mechanical tension generated by myosin II‐mediated contractile forces exerted on cell–matrix adhesions. When exposed to force, mechanosensitive adhesome proteins can change their conformation or expose cryptic‐binding sites leading to the recruitment of proteins, rearrangement of the cytoskeleton, reinforcement of the adhesive site and signal transduction. Biophysical methods and proteomics revealed force ranges within the adhesome and cytoskeleton, and also force‐dependent changes in adhesome composition. In this review, we provide an overview of the compositional dynamics of cell–matrix adhesions, discuss the most prevalent functional domains in adhesome proteins and review literature and concepts about mechanosensing mechanisms that operate at the adhesion site.

Structural Model of a CRISPR RNA-Silencing Complex Reveals the RNA-Target Cleavage Activity in Cmr4

The Cmr complex is an RNA-guided endonuclease that cleaves foreign RNA targets as part of the CRISPR prokaryotic defense system. We investigated the molecular architecture of the P. furiosus Cmr complex using an integrative structural biology approach. We determined crystal structures of P. furiosus Cmr1, Cmr2, Cmr4, and Cmr6 and combined them with known structural information to interpret the cryo-EM map of the complex. To support structure determination, we obtained residue-specific interaction data using protein crosslinking and mass spectrometry. The resulting pseudoatomic model reveals how the superhelical backbone of the complex is defined by the polymerizing principles of Cmr4 and Cmr5 and how it is capped at the extremities by proteins of similar folds. The inner surface of the superhelix exposes conserved residues of Cmr4 that we show are required for target-cleavage activity. The structural and biochemical data thus identify Cmr4 as the conserved endoribonuclease of the Cmr complex.

Conformational control of integrin subtype selectivity in isoDGR peptide motifs: A biological switch

Thumbnail image of graphical abstract The rearrangement of asparagine to isoaspartate (isoD) is responsible for the deactivation of many functional proteins. However, the isoDGR motif, which is optimally presented as a conformationally controlled cyclic pentapeptide, binds selectively to a5s1 integrin (see the docking model) with an affinity comparable to that of the peptidic antitumor agent Cilengitide

The kindlins at a glance.

The kindlin family of focal adhesion (FA) proteins consists of three evolutionarily conserved proteins, kindlin-1, -2 and -3 [also known as fermitin family homolog 1, 2 and 3 (FERM1, FERM2 and URP2), respectively], and was named after a rare congenital skin disease – the Kindler syndrome – which

Time- and compartment-resolved proteome profiling of the extracellular niche in lung injury and repair

The extracellular matrix (ECM) is a key regulator of tissue morphogenesis and repair. However, its composition and architecture are not well characterized. Here, we monitor remodeling of the extracellular niche in tissue repair in the bleomycin‐induced lung injury mouse model. Mass spectrometry quantified 8,366 proteins from total tissue and bronchoalveolar lavage fluid (BALF) over the course of 8 weeks, surveying tissue composition from the onset of inflammation and fibrosis to its full recovery. Combined analysis of proteome, secretome, and transcriptome highlighted post‐transcriptional events during tissue fibrogenesis and defined the composition of airway epithelial lining fluid. To comprehensively characterize the ECM, we developed a quantitative detergent solubility profiling (QDSP) method, which identified Emilin‐2 and collagen‐XXVIII as novel constituents of the provisional repair matrix. QDSP revealed which secreted proteins interact with the ECM, and showed drastically altered association of morphogens to the insoluble matrix upon injury. Thus, our proteomic systems biology study assigns proteins to tissue compartments and uncovers their dynamic regulation upon lung injury and repair, potentially contributing to the development of anti‐fibrotic strategies.

Mannose 6-Phosphate/Insulin-like Growth Factor 2 Receptor Limits Cell Invasion by Controlling αVβ3 Integrin Expression and Proteolytic Processing of Urokinase-type Plasminogen Activator Receptor

The multifunctional mannose 6-phosphate/insulin-like growth factor 2 receptor (M6P/IGF2R) is considered a tumor suppressor. We report here that RNA interference with M6P/IGF2R expression in urokinase-type plasminogen activator (uPA)/urokinase-type plasminogen activator receptor (uPAR) expressing human cancer and endothelial cells resulted in increased pericellular plasminogen activation, cell adhesion, and higher invasive potential through matrigel. M6P/IGF2R silencing led also to the cell surface accumulation of urokinase and plasminogen and enhanced expression of alphaV integrins. Genetic rescue experiments and inhibitor studies revealed that the enhanced plasminogen activation was due to a direct effect of M6P/IGF2R on uPAR, whereas increased cell adhesion to vitronectin was dependent on alphaV integrin expression and not uPAR. Increased cell invasion of M6P/IGF2R knockdown cells was rescued by cosilencing both uPAR and alphaV integrin. Furthermore, we found that M6P/IGF2R expression accelerates the cleavage of uPAR. M6P/IGF2R silencing resulted in an increased ratio of full-length uPAR to the truncated D2D3 fragment, incapable of binding most uPAR ligands. We conclude that M6P/IGF2R controls cell invasion by regulating alphaV integrin expression and by accelerating uPAR cleavage, leading to the loss of the urokinase/vitronectin/integrin-binding site on uPAR.

TGF-beta-induced apoptosis in endothelial cells mediated by M6P/IGFII-R and mini-plasminogen.

Transforming growth factor-β (TGF-β), a key modulator of endothelial cell apoptosis, must be activated from the latent form (LTGF-β) to induce biological responses. In the present study, we report activation of TGF-β by functional and physical co-operation of the mannose-6-phosphate/insulin-like-growth-factor-II receptor (CD222) and the urokinase-type plasminogen activator receptor (CD87). We show that endothelial cells express CD222 and CD87 in a membrane complex and demonstrate that the association of these two receptors is essential for the release of active TGF-β in the transduced mouse fibroblast used as model cells. By contrast, smooth-muscle cells, which express CD222 and CD87 at similar density to endothelial cells but not in complexed form, do not activate TGF-β. We also have found that mini-plasminogen is a high-affinity ligand for CD222 and is essential for the activation of TGF-β by the CD87-CD222 complex to induce apoptosis in endothelial cells. This specific mechanism of TGF-β-mediated apoptosis in endothelial cells is thus a potential novel target to be considered for treatment of pathological vascular disorders (e.g. tumor angiogenesis).

Sequential Cooperation of CD2 and CD48 in the Buildup of the Early TCR Signalosome

The buildup of TCR signaling microclusters containing adaptor proteins and kinases is prerequisite for T cell activation. One hallmark in this process is association of the TCR with lipid raft microdomains enriched in GPI-proteins that have potential to act as accessory molecules for TCR signaling. In this study, we show that GPI-anchored CD48 but not CD59 was recruited to the immobilized TCR/CD3 complex upon activation of T cells. CD48 reorganization was vital for T cell IL-2 production by mediating lateral association of the early signaling component linker for activated T cells (LAT) to the TCR/CD3 complex. Furthermore, we identified CD2 as an adaptor linking the Src protein tyrosine kinase Lck and the CD48/LAT complex to TCR/CD3: CD2 associated with TCR/CD3 upon T cell activation irrespective of CD48 expression, while association of CD48 and LAT with the TCR/CD3 complex depended on CD2. Consequently, our data indicate that CD2 and CD48 cooperate hierarchically in the buildup of the early TCR signalosome; CD2 functions as the master switch recruiting CD48 and Lck. CD48 in turn shuttles the transmembrane adapter molecule LAT.

Senolytic drugs target alveolar epithelial cell function and attenuate experimental lung fibrosis ex vivo

Idiopathic pulmonary fibrosis (IPF) is a devastating lung disease with poor prognosis and limited therapeutic options. The incidence of IPF increases with age, and ageing-related mechanisms such as cellular senescence have been proposed as pathogenic drivers. The lung alveolar epithelium represents a major site of tissue injury in IPF and senescence of this cell population is probably detrimental to lung repair. However, the potential pathomechanisms of alveolar epithelial cell senescence and the impact of senolytic drugs on senescent lung cells and fibrosis remain unknown. Here we demonstrate that lung epithelial cells exhibit increased P16 and P21 expression as well as senescence-associated β-galactosidase activity in experimental and human lung fibrosis tissue and primary cells. Primary fibrotic mouse alveolar epithelial type (AT)II cells secreted increased amounts of senescence-associated secretory phenotype (SASP) factors in vitro, as analysed using quantitative PCR, mass spectrometry and ELISA. Importantly, pharmacological clearance of senescent cells by induction of apoptosis in fibrotic ATII cells or ex vivo three-dimensional lung tissue cultures reduced SASP factors and extracellular matrix markers, while increasing alveolar epithelial markers. These data indicate that alveolar epithelial cell senescence contributes to lung fibrosis development and that senolytic drugs may be a viable therapeutic option for IPF. Alveolar epithelial cell senescence occurs in IPF and senolytic treatment attenuates experimental lung fibrosis http://ow.ly/nFlz30bsmNm